Opening the Channel: the Two Functional Interfaces of Pseudomonas aeruginosa OpmH with the Triclosan Efflux Pump TriABC

Abstract

TriABC-OpmH is an efflux pump from Pseudomonas aeruginosa with an unusual substrate specificity and protein composition. When overexpressed, this pump confers a high level of resistance to the biocide triclosan and the detergent SDS, which are commonly used in combinations for antimicrobial treatments. This activity requires an RND transporter (TriC), an outer membrane channel (OpmH), and two periplasmic membrane fusion proteins (TriA and TriB) with nonequivalent functions. In the active complex, TriA is responsible for the recruitment of OpmH, while TriB is responsible for stimulation of the transporter TriC. Here, we used the functional and structural differences between the two membrane fusion proteins to link their functional roles to specific interactions with OpmH. Our results provide evidence that the TriB-dependent stimulation of the TriC transporter is coupled to opening of the OpmH aperture through binding to the interprotomer groove of OpmH. IMPORTANCEMultidrug efflux transporters are important contributors to intrinsic and acquired antibiotic resistance in clinics. In Gram-negative bacteria, these transporters have a characteristic tripartite architecture spanning the entire two-membrane cell envelope. How such complexes are assembled and how the reactions separated in two different membranes are coupled to provide efficient efflux of various compounds across the cell envelope remain unclear. This study addressed these questions, and the results suggest a mechanism for functional integration of drug efflux by the inner membrane transporter and opening of the channel for transport across the outer membrane. In Gram-negative bacteria, polyspecific transporters detoxify the inner membrane and periplasm of noxious compounds and contribute to clinical antibiotic resistance (1). A characteristic structural feature of these transporters is the formation of tripartite complexes spanning both the inner and outer membranes of Gram-negative cell envelopes. Located in the outer membrane are proteins belonging to the outer membrane factor (OMF) family that act as channels for substrate expulsion across the low-permeability barrier of the outer membrane. OMFs show very little sequence similarity to one another but are structurally similar (2). OMF structures comprise both a ␤-barrel domain, a common structural feature of other outer membrane proteins, and a large ␣-helical barrel (3) (Fig. 1A). The periplasmic tip of the ␣-helical barrel converges to form a closed aperture that prevents leakage of the periplasmic content and uptake of various molecules from the external medium (Fig. 1B). Large extracellular loops on the external side of the ␤-barrel domain also help with blocking noxious chemicals from entering the cell. At the aperture, a series of ionic bridges act as the locking gate of the aperture and disruption of these causes a "leaky" phenotype (4, 5). A secondary gate of aspartate or, in a few cases, a ring of hydrophobic residues acts additionally to keep the aperture locked.In the resting state,...

An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump

“…The membrane-proximal (MP) domain of AcrA interacts with both the docking domain and the top of the pore domain of AcrB. Each AcrB protomer makes two non-equivalent interactions with AcrA ( Ntreh et al, 2016 ); i.e. each pair of AcrA protomers makes two conformationally distinct interfaces with AcrB, as was found with the interaction between AcrA and TolC ( Figures 4 and 5 ).…”

Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

“…On the example of the TriABC-OpmH system it has been established that PAP1 (TriA) is detrimental to recruitment of the OMF and hence likely makes first contact with it, which is supported by mutagenesis, 802 , 803 while the MPD of the PAP2 (TriB) is responsible for the activation of the transporter, which, in light of the latest findings of Glavier et al, 270 is likely mediated via the conformational changes that propagate to the “exit-gate”.…”

Trans-envelope multidrug efflux pumps of Gram-negative bacteria and their synergism with the outer membrane barrier

“…Importantly, each protomer in the MFP dimer binds to a specific binding site on OMF, and the two binding sites are not functionally equivalent [26, 31]. Apparently, one MFP subunit grasps OMF, whereas another is responsible for opening of the channel [40, 41]. Opening and closing of the channel could also be part of the trans-envelope transport mechanism that does not require disengagement of the complex [30].…”